沙眼衣原体感染后HeLa-229细胞的miRNA差异表达及其功能研究

发布时间：2018-03-06 04:07

本文选题：miRNA　切入点：沙眼衣原体　出处：《天津医科大学》2007年硕士论文　论文类型：学位论文

【摘要】：[目的]miRNA是近些年来新发现的一类能够调控其靶基因表达水平的小分子RNA家族。目前,1miRNA的功能研究主要集中于生长发育、肿瘤发生、细胞增殖、细胞凋亡等方面,随着研究的不断深入,发现1miRNA参与了病毒和细菌这些病原微生物与宿主细胞的相互作用过程。本研究的初衷在于进一步探索：miRNA在微生物感染宿主细胞过程中所发挥的作用及其调控机制,拓展我们关于miRNA功能的认识。 [方法]首先在本实验室建立沙眼衣原体的体外培养模型,使用HeLa-229作为宿主细胞培养扩增沙眼衣原体。并通过方法的改进提高了衣原体感染宿主细胞的效率,使绝大多数细胞都被沙眼衣原体感染,为之后的miRNA表达谱芯片实验提供更好的基础,而后使用miRNA表达谱芯片检测沙眼衣原体感染宿主细胞后,细胞中各个miRNA的表达水平的变化。从中筛选表达水平变化最为显著的一些,并结合已有的关于这些miRNA的研究进展进行综合分析,选取一些最有可能与衣原体感染相关的miRNA作为后续研究的候选者。最后针对这些候选的miRNA,根据其参与的生长调控,凋亡等不同机制,在衣原体感染这个模型的基础上,通过ASO技术封闭其功能；而后选择适当的实验对一些细胞生物学指标,如细胞生长曲线、Hoechst法凋亡检测等指标进行检测,以此来评估这止匕miRNA在衣原体感染过程中所发挥的作用。 [结果]1.成功建立了E血清型沙眼衣原体的体外细胞感染模型,并成功的提高了其感染效率。 2.通过miRNA表达谱芯片这种高通量的筛选方法筛选得到了14个表达水平明显变化的miRNA。其中9个在感染衣原体的细胞中表达水平明显上调,5个表达水平明显下调。 3.根据1miRNA表达谱芯片的结果提示,发现通过人为的封闭表达水平上升的9个niRNA的功能,使得沙眼衣原体在感染这种细胞后最终复制产生的子代衣原体明显减少；通过生长曲线实验证明has-miR-190、has-miR-372的低表达可以明显的抑制HeLa-229细胞的分裂增殖。由此推断沙眼衣原体感染后通过改变这两个miRNA的表达水平对宿主细胞的分裂增殖活性产生影响,最后通过Hoechst凋亡检测证实了has-miR-15、has-miR-16同时参与了衣原体感染后所诱导的宿主细胞凋亡过程,并发挥着明显的作用。 [结论]沙眼衣原体感染宿主细胞后对宿主细胞中多个miRNA的表达水平产生影响,而这些miRNA在衣原体感染和复制中发挥着明显的作用,尤其是has-miR-190、has-miR-372、has-miR-15a、has-miR-16分别通过调节宿主细胞的增殖和凋亡在感染过程中发挥作用。这些发现为进一步研究miRNA对衣原体感染的影响、阐明衣原体的感染机理提供了稳定的感染模型和大量有意义的线索。也为开发新的抗衣原体感染的治疗手段提供了新的理论基础。
[Abstract]:[objective] miRNA is a new class of small molecular RNA family which can regulate the expression level of its target gene in recent years. At present, the functional studies of miRNA mainly focus on the aspects of growth and development, tumorigenesis, cell proliferation, apoptosis and so on. With the deepening of the research, It was found that 1 miRNA is involved in the interaction of viruses and bacteria with host cells. The purpose of this study is to further explore the role and regulatory mechanism of microbes in the process of infection with host cells. Expand our understanding of miRNA functionality. [methods] the culture model of chlamydia trachomatis in vitro was established in our laboratory. Chlamydia trachomatis was amplified by using HeLa-229 as host cell culture, and the efficiency of chlamydia trachomatis infection was improved by the improvement of the method. Most of the cells were infected with Chlamydia trachomatis, which provided a better basis for the later miRNA expression microarray experiment, and then the miRNA expression microarray was used to detect chlamydia trachomatis infection in host cells. Some of the most significant changes in the level of expression of miRNA were screened from the cells, and combined with the existing research progress on these miRNA to conduct a comprehensive analysis. Some of the most likely miRNA related to chlamydia infection were selected as candidates for further study. Finally, based on the model of chlamydia infection, according to the different mechanisms of growth regulation, apoptosis and other mechanisms involved in these candidate miRNAs, Some cell biological indexes, such as cell growth curve Hoechst method and apoptosis detection, were selected to evaluate the role of miRNA in the process of chlamydia infection. The model of E serotype chlamydia trachomatis infection in vitro was successfully established and its infection efficiency was improved successfully. 2. 2. Fourteen miRNAs with significantly different expression levels were obtained by high throughput screening with miRNA expression microarray, of which 9 were up-regulated and 5 down-regulated in chlamydia infection cells. 3.According to the results of 1miRNA expression microarray, it was found that the function of 9 niRNA, which increased the expression level of chlamydia trachomatis, significantly reduced the generation of chlamydia trachomatis after infection. The results of growth curve experiment showed that the low expression of has-miR-190-has-miR-372 could significantly inhibit the mitotic proliferation of HeLa-229 cells. It was inferred that by changing the expression level of these two miRNA after chlamydia trachomatis infection, the mitotic activity of host cells could be affected by the low expression of has-miR-190-miR-372. Finally, it was confirmed by Hoechst apoptosis detection that has-miR-15 has-miR-16 was involved in the apoptosis of host cells induced by chlamydia infection and played a significant role. [conclusion] Chlamydia trachomatis infection affects the expression of multiple miRNA in the host cells, and these miRNA play a significant role in the infection and replication of chlamydia trachomatis. In particular, has-miR-190C has-miR-372Hhas-miR-15aHS-miR-16 plays a role in the process of infection by regulating the proliferation and apoptosis of host cells, respectively. These findings are intended to further study the effect of miRNA on chlamydia infection. Clarifying the mechanism of chlamydia infection provides a stable infection model and a large number of meaningful clues, and provides a new theoretical basis for the development of new anti-chlamydia infection treatment.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R3416

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